Although heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic ...acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear. Here, we report that the transcriptional regulator heme-regulated transporter regulator (HrtR) senses and binds a heme molecule as its physiological effector to regulate the expression of the heme-efflux system responsible for heme homeostasis in L. lactis. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates gene expression for the heme efflux system, we determined the crystal structures of the apo-HrtR·DNA complex, apo-HrtR, and holo-HrtR at a resolution of 2.0, 3.1, and 1.9 Å, respectively. These structures revealed that HrtR is a member of the TetR family of transcriptional regulators. The residue pair Arg-46 and Tyr-50 plays a crucial role for specific DNA binding through hydrogen bonding and a CH-π interaction with the DNA bases. HrtR adopts a unique mechanism for its functional regulation upon heme sensing. Heme binding to HrtR causes a coil-to-helix transition of the α4 helix in the heme-sensing domain, which triggers a structural change of HrtR, causing it to dissociate from the target DNA for derepression of the genes encoding the heme efflux system. HrtR uses a unique heme-sensing motif with bis-His (His-72 and His-149) ligation to the heme, which is essential for the coil-to-helix transition of the α4 helix upon heme sensing.
Background: A transcriptional regulator HrtR regulates the expression of the heme efflux transporter.
Results: The crystal structures of HrtR in apo, holo, and DNA-bound forms were solved.
Conclusion: Heme sensing by HrtR induces a coil-to-helix transition to regulate DNA binding activity.
Significance: Elucidating the structure and function of HrtR is fundamental to understand the molecular mechanism of heme homeostasis.
Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic ...reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe
-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H
from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.
Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The ...nature of the ferryl heme—and whether it is an FeIV=O or FeIV‐OH species—is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Å and 1.50 Å crystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X‐ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron‐oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine‐tuning is linked to the functional need for proton delivery to the heme.
Enzymatic fine‐tuning of heme reactivity: Free‐electron laser crystal structures of two different heme‐containing peroxidases—cytochrome c peroxidase and ascorbate peroxidase—show differences in the nature of the ferryl species. Precise enzymatic fine‐tuning within structurally similar heme active sites is implicated.
Nitric oxide (NO) plays diverse and significant roles in biological processes despite its cytotoxicity, raising the question of how biological systems control the action of NO to minimize its ...cytotoxicity in cells. As a great example of such a system, we found a possibility that NO-generating nitrite reductase (NiR) forms a complex with NO-decomposing membrane-integrated NO reductase (NOR) to efficiently capture NO immediately after its production by NiR in anaerobic nitrate respiration called denitrification. The 3.2-Å resolution structure of the complex of one NiR functional homodimer and two NOR molecules provides an idea of how these enzymes interact in cells, while the structure may not reflect the one in cells due to the membrane topology. Subsequent all-atom molecular dynamics (MD) simulations of the enzyme complexmodel in amembrane and structure-guided mutagenesis suggested that a few interenzyme salt bridges and coulombic interactions of NiR with the membrane could stabilize the complex of one NiR homodimer and one NOR molecule and contribute to rapid NO decomposition in cells. The MD trajectories of the NO diffusion in the NiR:NOR complex with the membrane showed that, as a plausible NO transfer mechanism, NO released from NiR rapidly migrates into the membrane, then binds to NOR. These results help us understand the mechanism of the cellular control of the action of cytotoxic NO.
Cytoglobin (Cygb) is a member of the hexacoordinated globin protein family and is expressed ubiquitously in rat and human tissues. Although Cygb is reportedly upregulated under hypoxic conditions ...both in vivo and in vitro, suggesting a physiological function to protect cells under hypoxic/ischemic conditions by scavenging reactive oxygen species or by signal transduction, the mechanisms associated with this function have not been fully elucidated. Recent studies comparing Cygbs among several species suggest that mammalian Cygbs show a distinctly longer C-terminal domain potentially involved in unique physiological functions. In this study, we prepared human Cygb mutants (ΔC, ΔN, and ΔNC) with either one or both terminal domains truncated and investigated the enzymatic functions and structural features by spectroscopic methods. Evaluation of the superoxide-scavenging activity between Cygb variants showed that the ΔC and ΔNC mutants exhibited slightly higher activity involving superoxide scavenging as compared with wild-type Cygb. Subsequent experiments involving ligand titration, flash photolysis, and resonance Raman spectroscopic studies suggested that the truncation of the C- and N-terminal domains resulted in less effective to dissociation constants and binding rates for carbon monoxide, respectively. Furthermore, structural stability was assessed by guanidine hydrochloride and revealed that the C-terminal domain might play a vital role in improving structure, whereas the N-terminal domain did not exert a similar effect. These findings indicated that long terminal domains could be important not only in regulating enzymatic activity but also for structural stability, and that the domains might be relevant to other hypothesized physiological functions for Cygb.
N and C-terminal domains of human cytoglobin (Cygb) were explored to clarify physiological functions. Although both domains of Cygb showed no significant effect in binding of exogenous ligand; C-terminal domain, peculiar to mammalian Cygbs, contributed to regulate superoxide-scavenging activity slightly and to improve structural stability significantly. Display omitted
•Truncation of terminal domains slightly enhances superoxide-scavenging activity.•Terminal domains slightly influence in monomer with an intramolecular disulfide bond.•C-terminal domain peculiar to mammalian is involved in structural stability.•Terminal domains might play important roles for protein-protein interactions.
Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These ...bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction.
Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R–CH=N–OH) to their corresponding nitrile (R–C▪N). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its ...physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 Å resolution and the n-butyraldoxime- and propionaldoxime-bound OxdREs at 1.8 and 1.6 Å resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe3+ heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.
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